Covalent attachment of the fourth component of complement to the immune complex (IC) is a critical events in the classical pathway. This event not only focuses assembly of the later components at the site of inflammation but is though to be important in inhibiting the formation of insoluble complexes. Two isotypes of C4 protein are found in the serum of most individuals and through their structures are nearly identifical, they differ significantly in their efficiency of covalent binding to IC. While C4A selectively forms an amide linkage with protein, C4B more readily forms an ester bond with carbohydrate. It has been proposed that the selectively in binding evolved as a mechanism to insure attachment of C4 to a wide range of pathogens or structurally different forms of antibody. Genetic studies have shown that there is an increased relative risk to a number of autoimmune diseases, particularly those complicated by immune complex deposition such as SLE, with certain C4 allotypes. The association is greatest with partial and complete deficiencies of C4 protein. The objective of this proposal is to determine the chemical basis for the binding differences between the two isotypes of C4 protein. This will be accomplished by analysing hybrid C4 proteins constructed by the technique of site-directed mutagenesis and includes: (1) determining which amino acid residues are critical for the selectivity in transacylation of C4 to either protein or carbohydrate; (2) determining what role allotypic differences have in the interaction and covalent binding of C4 to antigen and antibody; (3) determining what role carbohydrate has in covalent binding; (4) determining which residues are critical for stability of the thiolester.